Finessing, Extending and Developing an Overview of the Regulation of Ascorbate in plants (FEDORA)
Lead Research Organisation:
University of Exeter
Department Name: Biosciences
Abstract
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Technical Summary
FEDORA aims to integrate understanding of the regulation of ascorbate in plants.
1) We will define the molecular mechanism of negative feedback regulation of GGP translation using 5'uORF:luciferase reporters to identify the signal in plant cell and algal cultures.
2) We will use wheat germ in vitro translation to investigate the mechanisms of uORF translation and ribosome stalling that impair translation of GGP using Ribo-seq and modified ChIP to define the association between the uORF peptide and the stalled ribosome(s), leading to structural (low) resolution of the complex.
3) We will investigate how translational control of GGP is set within the boundaries defined by transcriptional control of GGP expression, by comparing proGGP:luciferase reporters with and without mutated uORFs in tomato and Arabidopsis.
4) We will use Arabidopsis lines expressing the proGGP muORF:luciferase reporters to establish a genetic screen for mutations affecting transcriptional regulators of GGP in Arabidopsis.
5) We will test whether transcriptional control by regulators already identified in tomato also operates in Arabidopsis, using T-DNA insertion mutants. We will use genome editing in tomato to develop required mutations in MoneyMaker genetic background.
6) We will investigate how the control of GGP activity impacts flux to ascorbate using targeted MRM analysis by LC-QQQ MS/MS.
7) We will investigate the physiological roles of ascorbate in different plant species by defining the phenotypic consequences of a series of GGP alleles with mis-regulated ascorbate levels, measuring ascorbate and glutathione, leaf number, leaf shape, flowering time, flower number, floral morphology/organ fusion, root biomass, fruit set, fruit weight, shelf life and resistance to necrotrophic pathogens,
8) We will determine whether similar or different phenotypes are observed in Arabidopsis by creating muORFAtGGP1/VTC2 and muORFAtGGP2/VTC5 double mutants by genome editing of Arabidopsis.
1) We will define the molecular mechanism of negative feedback regulation of GGP translation using 5'uORF:luciferase reporters to identify the signal in plant cell and algal cultures.
2) We will use wheat germ in vitro translation to investigate the mechanisms of uORF translation and ribosome stalling that impair translation of GGP using Ribo-seq and modified ChIP to define the association between the uORF peptide and the stalled ribosome(s), leading to structural (low) resolution of the complex.
3) We will investigate how translational control of GGP is set within the boundaries defined by transcriptional control of GGP expression, by comparing proGGP:luciferase reporters with and without mutated uORFs in tomato and Arabidopsis.
4) We will use Arabidopsis lines expressing the proGGP muORF:luciferase reporters to establish a genetic screen for mutations affecting transcriptional regulators of GGP in Arabidopsis.
5) We will test whether transcriptional control by regulators already identified in tomato also operates in Arabidopsis, using T-DNA insertion mutants. We will use genome editing in tomato to develop required mutations in MoneyMaker genetic background.
6) We will investigate how the control of GGP activity impacts flux to ascorbate using targeted MRM analysis by LC-QQQ MS/MS.
7) We will investigate the physiological roles of ascorbate in different plant species by defining the phenotypic consequences of a series of GGP alleles with mis-regulated ascorbate levels, measuring ascorbate and glutathione, leaf number, leaf shape, flowering time, flower number, floral morphology/organ fusion, root biomass, fruit set, fruit weight, shelf life and resistance to necrotrophic pathogens,
8) We will determine whether similar or different phenotypes are observed in Arabidopsis by creating muORFAtGGP1/VTC2 and muORFAtGGP2/VTC5 double mutants by genome editing of Arabidopsis.